WO1996041640A1 - Analogues de la bradykinine utilises comme inhibiteurs selectifs de la thrombine - Google Patents

Analogues de la bradykinine utilises comme inhibiteurs selectifs de la thrombine Download PDF

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WO1996041640A1
WO1996041640A1 PCT/US1996/009940 US9609940W WO9641640A1 WO 1996041640 A1 WO1996041640 A1 WO 1996041640A1 US 9609940 W US9609940 W US 9609940W WO 9641640 A1 WO9641640 A1 WO 9641640A1
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pro
peptide
arg
seq
gly
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PCT/US1996/009940
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English (en)
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Alvin H. Schmaier
Ahmed A. K. Hasan
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The Regents Of The University Of Michigan
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Priority to CA002221865A priority Critical patent/CA2221865C/fr
Priority to DE69627191T priority patent/DE69627191T2/de
Priority to AT96923268T priority patent/ATE235912T1/de
Priority to US08/676,242 priority patent/US6143719A/en
Priority to AU63828/96A priority patent/AU703256B2/en
Priority to EP96923268A priority patent/EP0871464B1/fr
Priority to JP50324397A priority patent/JP4067117B2/ja
Publication of WO1996041640A1 publication Critical patent/WO1996041640A1/fr
Priority to HK99101383A priority patent/HK1016090A1/xx

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides

Definitions

  • This invention relates to the inhibition of ⁇ -thrombin-induced and ADP-induced cell activation.
  • Bradykinin is a vasoactive peptide released from the precursor plasma kininogens by kallikrein and other enzymes (Silva et al. , Amer. J. Physiol. 156: 261-274 (1949)). Bradykinin has been described to have multiple physiologic functions, including the stimulation of prostacyclin production (Hong, S.L., Thromb. Res. 18, 787 (1980); Crutchley et al , Biochim Biophy Ada 751, 99 (1983)) and the stimulation of the release of plasminogen activators (Smith et al , Blood 66, 835 (1983)).
  • Bradykinin induces superoxide formation and endothelium-dependent smooth muscle hyperpolarization (Holland, J.A. et al , J. Cell Physiol. 143, 21 (1990); Nakashima, M. et al , J. Clin. Invest. 92, 2867 (1993)).
  • bradykinin is the major inducer of nitric oxide formation (Palmer, R.M.J. et al. , Nature 327, 524 (1987)). Bradykinin has been characterized to produce vasodilation in most vascular beds which in the coronary artery circulation results in increased blood flow (Line et al , J. Mol. Cell Cardiol. 24, 909 (1992)).
  • bradykinin as a cardioprotective agent (Line et al , supra; Gohlke et al , Hypertension 23, 411 (1994); Parratt et al , Cardiovascular Research 28, 183 (1994); Zanzinger et al. , Cardiovascular Research 28, 209 (1994)). Bradykinin's elevation by angiotensin converting enzyme inhibitors is believed to be the mechanism by which these drugs promote their beneficial effects on heart failure.
  • bradykinin In addition to the delivery of bradykinin, its parent proteins, high
  • HK and low (LK) molecular weight kininogens also have the ability to be selective inhibitors of ⁇ -thrombm, inhibiting ⁇ -thrombin's ability to activate cells without interfering with its enzymatic ability (Meloni et al. , J. Biol. Chem. 266, 6786 (1991); Puri et al , Blood 11, 500 (1991)). This activity was believed to be a unique function for the kininogens; one which had not been ascribed to other proteins. Most naturally occurring human protein inhibitors of ⁇ -thrombin are directed towards its protease activity.
  • HK and LK are selective inhibitors of thrombin's ability to activate platelets by blocking ⁇ -thrombin from binding to the platelet membrane (Meloni et al, supra; Puri et al, supra). This activity of the kininogens appeared to be localized to domain 3 on their heavy chain since isolated domain 3 retains that activity (Jiang et al, J. Biol. Chem. 267, 3712 (1992)).
  • Inhibition of platelet activation by domain 3 is observed by a marked decrease in the platelet' s ability to aggregate and secrete their granule contents.
  • the granule contents comprise proteins which participate in hemostasis, thrombosis, and the inflammatory response.
  • Inhibition of endothelial cell activation may similarly be observed by a decrease in secretion of endothelial cell contents such as tissue plasminogen activator and von Willebrand factor.
  • the domain 3 polypeptide like its parent proteins HK and LK functions to inhibit cell activation by blocking thrombin binding to its target cells. This polypeptide is a selective inhibitor of thrombin-induced platelet activation.
  • Administration of domain 3 therefore does not impact on induction of platelet * activation by physiological substances other than thrombin, such as, for example collagen, adenosine diphosphate, epinephrine and platelet activating factor.
  • antiplatelet agents Two competing classes of antiplatelet agents for the prevention of coronary thrombosis are being considered.
  • One class of agents including monoclonal antibody 7E3, aims to block the final common pathway of platelet activation by inhibiting glycoprotein Ilb/IIIa (GPIIb/IIIa), integrin ⁇ , Ib ⁇ 3 . 7E3 is an effective agent, but it is a murine antibody and is antigenic in humans.
  • a second class of antiplatelet agents inhibit a presumed, primary initiating agent of platelet activation, ⁇ -thrombin.
  • Phe-Pro-Arg-chloromethylketone a potent inhibitor of ⁇ -thrombin' s proteolytic activity, prolongs the bleeding time, a crude measure of platelet function (Hanson, S.R. et al, Proc. Natl. Acad. Sci. 85, 3184-3188 (1988)).
  • the first generation of potent ⁇ -thrombin proteolytic inhibitors to enter into clinical trials is a recombinant product derived from medicinal leeches, hirudin. This compound, which is a small molecular mass and is not considered to be antigenic, is a potent anti-thrombin.
  • a synthetic analog of hirudin, hirulog combines the anion exosite I binding properties of hirudin with the proteolytic inhibitory activity of PPACK.
  • both drugs were effective inhibitors of platelet activation; however, the tradeoff for effective anticoagulation was increased hemorrhage leading to the termination of three clinical trials.
  • non-selective proteolytic inhibitors of a- thrombin are not clinically tolerated and may never have commercial significance.
  • An ideal anti-thrombotic to prevent arterial thrombosis would be one which prevents platelet and endothelial cell activation without preventing the proteolytic activity of ⁇ -thrombin to clot fibrinogen and activate protein C, factor XIII, and factors V and VIII.
  • HK and LK high molecular weight kininogens
  • HK and LK low molecular weight kininogens
  • Both low and high molecular weight kininogens have identical amino acid sequences from their amino-terminus through 12 amino acids beyond the carboxy-terminus of bradykinin.
  • LK and HK share a common heavy chain (62 kDa), the bradykinin (BK) moiety (0.9 kDa), and the first 12 amino acids of the amino terminal portion of each of their "light chains” (Takagaki, Y. et al, J. Biol Chem. 260:8601-8609 (1985); Kitamura, N. et al, J. Biol Chem., 260:8610-8617 (1985)).
  • This identity corresponds to residues 1 through about residue 383.
  • human kininogen shall mean, unless otherwise indicated, both high and low molecular weight forms of any kininogen molecule, in all its various forms derived from human plasma, platelets, endothelial cells, granulocytes, or skin or other tissues or organs, regardless of whether it is found in the fluid or the tissue phase.
  • Light chain shall mean, when referring or relating to human kininogen, the 56 kDa intermediate plasma kallikrein-cleavage fragment of HK which has the ability to correct the coagulant defect in total kininogen-deficient plasma.
  • Heavy chain shall mean, when referring or relating to human kininogen, the 64 kDa kallikrein-cleavage fragment of HK or LK, which is free of bradykinin and "light chain”.
  • Domain 3 with respect to the kininogen heavy chain shall mean the trypsin-cleavage fragment of the human kininogen heavy chain which is about 21 kDa.
  • natural amino acid is meant any of the twenty primary, naturally occurring amino acids which typically form peptides and polypeptides.
  • synthetic amino acid is meant any other amino acid, regardless of whether it is prepared synthetically or derived from a natural source.
  • BK analog is meant a peptide having an amino acid sequence analogous to the sequence of the nonapeptide bradykinin, which is capable of inhibiting ⁇ -thrombin from cleaving its receptor on platelets and other cells, such that the peptide prevents the alteration or loss of the SPAN 12 epitope on the thrombin receptor, and blocks cleavage of a peptide, NAT12 (SEQ ID NO:2), which spans the ⁇ -thrombin cleavage site on the thrombin receptor. BK analogs are thus able to inhibit thrombin-induced platelet activation.
  • ATAP 138 monoclonal antibody specific for an epitope on the thrombin receptor, which epitope is preserved following ⁇ -thrombin cleavage of the receptor BK: bradykinin D3: domain 3 of kininogen DFP: diisopropyl fluorophosphate
  • D-Tic D- 1 ,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl
  • EDTA ethylenediaminetetraacetic acid
  • HBTU 2-(l -H-benzotriazole- 1 - YL)- 1 , 1 ,3,3-tetramethyl- uroniumhexofluorophosphate HOBt 1-hydroxybenzotriazole
  • LK human low molecular weight kininogen
  • NAT12 peptide sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-
  • Leu-Leu-Arg which spans the ⁇ -thrombin cleavage site on the thrombin receptor Oic: (3a5, 7a5)-octahydroindol-2-yl-carbonyl
  • PADGEM platelet activation dependent granule external membrane protein, also known as P-selectin, GMP140 or CD62 PGE 1 : prostaglandin E 1
  • SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis SPAN 12 monoclonal antibody specific for the sequence Asn- Ala-Thr-
  • TRAP thrombin receptor activation peptide. which has the amino acid sequence Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID NO: 19)
  • Tris tris(hydroxymethy l)aminomethane Summary of the Invention
  • the invention comprises a method of inhibiting thrombin-induced platelet or other cell activation comprising administering to an individual in need of such treatment an effective amount of a peptide which inhibits thrombin activation of platelets or other cells, wherein said peptide has an amino acid sequence of the formula:
  • X is from zero to thirty natural or synthetic amino acids; and X 2 is from zero to thirty natural or synthetic amino acids; provided that the peptide may not be native bradykinin.
  • X is zero to seven amino acids and X 2 is zero to nine amino acids.
  • the peptide according to formula I has the sequence Arg-Pro-Pro-Gly- Phe (SEQ ID NO:20).
  • the invention further comprises a method for inhibiting ADP- induced platelet activation, which method comprises administering to an individual in need of such treatment an effective amount of a peptide according to formula I.
  • Another embodiment of the invention comprises a method for preventing platelet aggregation comprising administering to an individual in need of such treatment an effective amount of a peptide according to formula I.
  • a method of inhibiting ADP-induced platelet activation comprises administering to an individual in need of such treatment an effective amount of a peptide, which inhibits thrombin activation of platelets or other cells, wherein said peptide is comprised of one or more segments having the amino acid sequence X,-Arg-Pro- Pro-Gly-X 2 and the peptide has the formula:
  • L is a linker comprising a covalent bond or chemical group
  • X123 which may be the same or different in each segment, is from zero to thirty natural or synthetic amino acids
  • X 2 which may be the same or different in each segment, is from zero to thirty natural or synthetic amino acids
  • n is an integer from two to twenty.
  • the segment of a peptide according to formula II has the sequence Arg-Pro-Pro-Gly-Phe (SEQ ID NO:20).
  • the invention further comprises a method for inhibiting ADP- induced platelet activation, which method comprises administering to an individual in need of such treatment an effective amount of a peptide according to formula II, wherein L, X,, X 2 , and n are defined as above.
  • Another embodiment of the invention comprises a method for preventing platelet aggregation comprising administering to an individual in need of such treatment an effective amount of a peptide according to formula II, wherein L, X,, X 2 , and n are defined as above.
  • the invention as described herein also comprises a compound having the formula: Arg-Pro-Pro-Gly-Phe-Glu
  • Figures 1A-1D are plots of the inhibition of ⁇ -thrombin-induced platelet aggregation and secretion by BK (1A) and BK analogs (IB: SEQ ID NO:14, 1C: SEQ ID NO:13; ID: SEQ ID NO:18), incubated in the absence or presence of increasing concentrations of peptides before the addition of human ⁇ - thrombin to start the reaction: % residual aggregation activity ( O ); % residual [ 14 C]5-hydroxytryptamine secretion (D). Each figure is the mean ⁇ SEM of the data derived from at least three experiments.
  • Figures 2A-2D are plots of ⁇ -thrombin-induced calcium mobilization in human platelets in the presence of ⁇ -thrombin alone (2A), HK (2B); BK (SEQ ID NO:l) (2C); or BK analog SEQ ID NO: 14 (2D). Each figure is a representative experiment from at least three experiments.
  • Figure 3 is a plot of the inhibition of ⁇ -thrombin mediated calcium mobilization by BK (SEQ ID NO:l) and BK analog SEQ ID NO: 14. Increasing concentrations (0.01 mM to 2 mM) of BK (D) or SEQ ID NO: 14 ( O) were incubated with gel filtered platelets before the addition of ⁇ -thrombin. The data was plotted as the percent inhibition of Ca 2+ mobilized in the peptide-treated samples versus an untreated sample. The Figure is the mean ⁇ SEM of the data derived from three identical experiments at each concentration.
  • Figures 4A-4D are plots of the influence of BK analog SEQ ID NO:
  • FIG. 5 is a plot of the inhibition of I25 I- ⁇ -thrombin binding to platelets in the absence (G) or presence of 200 nM HK ( O), 1 mM of the BK analog SEQ ID NO:14 (O), or 1 mM of the BK analog SEQ ID NO:8 ( ⁇ ).
  • the Figure is the mean ⁇ SEM of the data derived from three experiments.
  • Figures 6A-6F are flow cytograms showing the effect of various BK analogs on expression of the antigenicity of the thrombin receptor. Washed platelets were incubated with monoclonal antibody SPAN 12 alone (Fig. 6A) or in the presence of 1 mM of BK (Fig. 6B), SEQ ID NO: 14 (Fig. 6C), SEQ ID NO: 18 (Fig. 6D) or SEQ ID NO:4 (Fig. 6E). The ghost curves represent unstimulated platelets; the solid curves represent ⁇ -thrombin activated platelets. Mouse IgG "(Fig. 6F) was used as a control. Each figure is a representative experiment of three experiments.
  • Figures 7A-7D are flow cytograms showing the influence of BK analog SEQ ID NO: 14 on the binding of monoclonal antibody ATAP138 to the thrombin receptor after ⁇ -thrombin activation of platelets (Fig. 7B). Control experiments were also performed with mouse IgG (Fig. 7C) and an antibody to CD62 (Fig. 7D). The ghost curves represent thrombin receptor expression by unstimulated platelets; the solid curves represent expression by ⁇ -thrombin activated platelets. The flow cytograms of Figures 7A-7D were performed on the same day with the same platelets as the flow cytograms in Figures 6A-6D. Each figure is a representative experiment of three experiments.
  • Figures 8A-8F are chromatographs showing the influence of BK analog SEQ ID NO:20 and a non-BK analog peptide (SEQ ID NO:22) on ⁇ - thrombin-induced cleavage of the thrombin receptor peptide NAT 12 (SEQ ID NO:2).
  • NAT12 SEQ ID NO:2 was incubated in the absence (Fig. 8A) or presence of ⁇ -thrombin (Fig. 8C).
  • NAT12 (SEQ ID NO:2) was incubated with ⁇ -thrombin in the absence (Fig. 8C) or presence of BK analog SEQ ID NO:20 (Fig. 8D).
  • Figure 8E is the chromatograph for NAT12 (SEQ ID NO:2) incubated with HK in the presence of ⁇ -thrombin
  • Figure 8F is the corresponding chromatograph for a non-BK analog peptide (SEQ ID NO:22).
  • Figure 9 is a plot of the plasma concentration of BK analog SEQ ID NO:
  • Figure 10 is a plot of the inhibition of thrombin- or ADP-induced rabbit platelet aggregation over time after a single infusion of BK analog SEQ ID NO:20.
  • Figure 11 is an aggregometer tracing of ⁇ -thrombin-induced (20 nM) aggregation of human platelets treated in the presence of 1 mM of a non-BK analog peptide (SEQ ID NO:22), 0.5 mM ofa BK analog heterodimer (“HETERODIMER”) (SEQ ID NO:22), 0.5 mM of 4-MAP, and 1 mM of a BK analog SEQ ID NO:20, and ⁇ -thrombin alone (control).
  • SEQ ID NO:22 non-BK analog peptide
  • HETERODIMER BK analog heterodimer
  • 4-MAP 0.5 mM of 4-MAP
  • the invention is directed to a method for preventing thrombosis by the use of bradykinin sequence-related analogous peptides that act as selective anti- thrombins.
  • the BK analogs are selective anti-thrombins because they are able to inhibit human ⁇ -thrombin and ⁇ -thrombin from activating platelets without interfering with ⁇ -thrombin's ability to proteolyze its various substrates, e.g., fibrinogen and factor V.
  • Most known thrombin inhibitors, hirudin, hirulog and PPACK interfere with ⁇ -thrombin's action by blocking all of its proteolytic activity.
  • BK analogs utilized in the present method would allow for inhibition of cell-induced plug formation without interfering with ⁇ -thrombin's enzymatic activity.
  • BK analogs may be used to prevent arterial occlusions arising from coronary thrombosis and stroke. We have found that the BK analogs inhibit thrombin from cleaving the thrombin receptor which is expressed on platelets.
  • the BK analogs have the ability to inhibit thrombin-induced platelet activation by blocking cleavage of the thrombin receptor and subsequent activation of platelets by exposure of the new amino terminus of the cleaved receptor.
  • Administration of a BK analog as described herein comprises a therapeutic method for inhibiting thrombin-induced activation of platelets, endothelial cells, brain cells, f ⁇ broblasts, smooth muscle cells, or other cells that contain a receptor for thrombin. This function inhibits platelet thrombus formation and other activities mediated by the thrombin receptor.
  • the BK analogs do not inhibit platelet activation by the same mechanism as intact kininogen and its isolated domain 3.
  • BK analogs do not inhibit 125 I- ⁇ -thrombin binding to platelets, as does a molar excess of purified HK, LK, or isolated domain 3.
  • BK analogs 1) block ⁇ -thrombin-induced calcium mobilization in platelets;
  • BK analogs act to inhibit platelet and other cell activation by inhibiting ⁇ -thrombin from cleaving its receptor on platelets and other cells.
  • the BK analog represents a chain truncation analog of a parent segment from the mature human kininogen heavy chain, which parent segment spans kininogen heavy chain amino acids 333 to 396, wherein the analog includes the core sequence Arg-Pro-Pro-Gly, which core sequence corresponds to kininogen heavy chain residues 363-366.
  • the BK analog represents a chain truncation analog of the kininogen heavy chain parent segment, which peptide contains the core sequence Arg-Pro-Pro-Gly, and up to 7 amino acids from the kininogen heavy chain parent segment upstream (in the amino terminus direction) of the core sequence, and up to 9 amino acids from the kininogen heavy chain parent segment downstream (in the carboxy terminus direction) of the core sequence. More preferably, the amino acids added to the amino terminus and the carboxy terminus of the core sequence are selected from kininogen heavy chain residues 357-363 and 367-383, respectively.
  • the amino acid sequence of the human kininogen heavy chain parent segment is given herein as SEQ ID NO:24. The complete sequence for human kininogen heavy chain can be found in Kellerman et al, Eur. J. Biochem. 154:471-478 (1986), the entire disclosure of which is incorporated herein by reference.
  • NH 3 + where R is a hydrogen atom or any organic group have been added to either the carboxyl or amino terminus of a peptide comprising the core sequence (Arg-Pro- Pro-Gly) (SEQ ID NO:21 ) of the native BK sequence segment (SEQ ID NO: 1 ) in order to form chain expansion analogs.
  • amino acids have been added to either the carboxyl or amino terminus of the five amino acid sequence, Arg-Pro- Pro-Gly-Phe (SEQ ID NO:20). Up to thirty amino acids may be added to either the carboxyl or amino terminus of the core sequence (SEQ ID NO:21) or BK analog SEQ ID NO:20.
  • the peptide comprises the amino acid sequence Arg-Pro-Pro-Gly-Phe (SEQ ID NO:20).
  • An example of the BK analogs included in this invention is the BK analog SEQ ID NO: 14 in which two amino acids have been added to the amino terminus and ten amino acids have been added to the carboxyl terminus of the core sequence, Arg- Pro-Pro-Gly (SEQ ID NO:21).
  • the peptide is HOE 140, having an amino acid sequence of (D-Arg)-Arg-Pro-Hyp-Gly-Thi-Ser-(D-Tic)-Oic- Arg (SEQ ID NO:17).
  • HOE140 may be purchased from Hoechst, Frankford, Germany or prepared according to the method of Hock et al. , Br. J. Pharmacol. 102:758-773 (1991) and Lambeck et al, Br. J. Pharmacol. 102:297-304 (1991), the entire disclosure of which is incorporated herein by reference.
  • two or more single-chain BK analogs are joined by one or more linkers, L, to form homodimers and heterodimers.
  • homodimers and heterodimers include dimers, trimers, and other multimers.
  • a homodimer is comprised of two or more identical single-chain BK analogs; heterodimers are comprised of two or more different single-chain BK analogs.
  • the linker can be either a covalent bond or a chemical group.
  • the number of single-chain BK analogs that can be joined is from two to thiry-two.
  • the number of BK analogs joined is from two to twenty, more preferably from two to eight, and most preferably, from two to four.
  • the BK analogs to be joined can be identical or they can be different.
  • An example of a covalent bond linking two single-chain BK analogs is the disulfide bond formed by the oxidation of two single chain BK analogs containing cysteine amino acids. This may require initially modifying the parent peptide so that the peptide includes a Cys residue in the appropriate position. Cysteine residues on single-chain BK analogs can be oxidized to form BK analog dimers by dissolving 1 mg of the single-chain peptide in 1.5 ml of 0.1% (v/v) 17.5 mM acetic acid, pH 8.4, followed by flushing with nitrogen and then 0.01 M K 2 Fe(CN) 6 . After incubation for one hour at room temperature, the dimer peptide is purified by HPLC.
  • a suitable covalent bond for linking two single- chain BK analogs is the amide bond formed by reacting the amino group of a lysine amino acid residue on one chain with the carboxylic acid group of a glutamic or aspartic amino acid residue of another chain.
  • the linking group can be formed by the covalent bond between two single-chain BK analogs using a cross-linking reagent.
  • homodimers and heterodimers can be prepared by first preparing S-(-7V- hexylsuccinimido)-modified peptide monomers according to the method of Cheronis et al, J Med. Chem. 37: 348 (1994).
  • N-hexylmaleimide a precursor for the modified peptide monomers, is prepared from N-(methoxycarbonyl)maleimide and N-hexylamine by mixing the two compounds in saturated ⁇ aHCO 3 at 0°C according to the procedure of Bodanszky and Bodanszky, The Practice of Peptide Synthesis; Springer-Verlag, New York, pp. 29-31 (1984). The product of the resulting reaction mixture is isolated by extraction into ethyl acetate, followed by washing with water, dried over Na 2 SO 4 , and is then concentrated in vacuo to produce N-hexylmaleimide as a light yellow oil.
  • S-(N-Hexylsuccinimido)- modified peptide monomers are then prepared from a cysteine-containing peptide (monomer) and ⁇ -hexylmaleimide by mixing one part peptide with 1.5 parts N- hexylmaleimide in dimethylformamide (3.3 ml/mM peptide) followed by addition to 30 volumes of 0.1 M ammonium bicarbonate, pH 7.5. The S-alkylation reaction carried out in this manner is complete in 30 min. The resulting S-(N- hexylsuccinimido)-modified peptide monomer is purified by preparative reverse- phase HPLC, followed by lyophilization as a fluffy, white powder.
  • Bissuccinimidohexane peptide dimers may be prepared according to the method of Cheronis et al. , supra from cysteine-substituted peptides in the same or different positions, respectively.
  • a mixture of one part bismaleimidohexane is made with two parts peptide monomer in dimethylformamide (3.3 ml/mM peptide) followed by addition to 0.1 ammonium bicarbonate, pH 7.5.
  • the reaction mixture is stirred at room temperature and is usually completed within 30 min.
  • the resulting bissuccinimidohexane peptide dimer is purified by preparative reverse-phase HPLC. After lyophilization the material is a fluffy, white powder.
  • Covalently cross-linked BK analog dimers of the present invention may be prepared by utilizing homobifunctional cross-linking reagents, e.g., disuccinimidyl tartrate, disuccinimidyl suberate, ethylene glycolbis(succinimidyl succinate), l,5-difluoro-2,4-dinitrobenzene ("DF ⁇ B”), 4,4'-diisothiocyano-2,2'- disulfonic acid stilbene (“DIDS”), and bismaleimidohexane (“BMH”).
  • the cross- linking reaction occurs randomly between the single-chain BK analogs.
  • heterobifunctional cross-linking reagents may be employed.
  • Such agents include, for example, N-succinimidyl-3-(2- pyridyldithio)propionate (“SPDP”), s ⁇ Jfosuccinirmdyl-2-( -azidosalicylamido)ethyl- l-3'-dithiopropionate ("SASD", Pierce Chemical Company, Rockford, IL), N- * maleimidobenzoyl-N-hydroxy-succinimidyl ester (“MBS”), m- maleimidobenzoylsulfosuccinimide ester (“sulfo-MBS”), N-succinimidyl(4- iodoacetyl)aminobenzoate (“SIAB”), succinimidyl 4-(N-maleimidomethyl)- cyclohexane- 1 -carboxylate (“SMCC”), succinimidyl-4-( ⁇ -maleirnidophenyl)butyrate
  • a first single-chain BK analog is derivatized with, e.g., the N-hydroxysuccinimidyl portion of the bifunctional reagent, and the derivatized BK analog is purified by gel filtration.
  • a second single-chain BK analog (which may or may not be the same or different from the first BK analog) is reacted with the second functional group of the bifunctional reagent, assuring a directed sequence of binding between components of the BK dimer.
  • Typical heterobifunctional cross-linking agents for forming protein- protein conjugates have an amino-reactive N-hydroxysuccinimide ester (NHS-ester) as one functional group and a sulfhydryl reactive group as the other functional group.
  • NHS-ester N-hydroxysuccinimide ester
  • epsilon-amino groups of surface lysine residues of the first single chain BK analog are acylated with the NHS-ester group of the cross-linking agent.
  • the second single chain BK analog, possessing free sulfhydryl groups is reacted with the sulfhydryl reactive group of the cross-linking agent to form a covalently cross-linked dimer.
  • Common thiol reactive groups include maleimides, pyridyl disulfides, and active halogens.
  • MBS contains a NHS-ester as the amino reactive group, and a maleimide moiety as the sulfhydryl reactive group.
  • Photoactive heterobifunctional cross-linking reagents e.g., photoreactive phenyl azides, may also be employed.
  • One such reagent, SASD may be linked to a single-chain BK analog via its NHS-ester group. The conjugation reaction is carried out at pH 7 at room temperature for about 10 minutes. Molar ratios between about 1 and about 20 of the cross-linking agent to the BK analog may be used.
  • the purified, derivatized BK analog is collected by affinity chromatography using a matrix having affinity for BK analogs, e.g., a polyclonal antibody reared to the BK analog.
  • Antibody for this purpose may be prepared by coupling the BK analog to key hole limpet hemocyanin using l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide-HCL (Goodfriend et ⁇ l, Science 144, 1344 (1964)).
  • the resulting conjugate is used to immunize rabbits by the procedure of M ⁇ ller-Esterl et ⁇ , Methods Enzymol 163, 240 (1988) to produce anti-BK analog antibodies.
  • the purified antibody is coupled to AFFIGEL 10 (Bio-Rad, Richmond, CA) to form an affinity column.
  • Immobilized anti-BK analog antibody, with the derivatized BK analog bound thereto, is then removed from the column by 0.2 M glycine elution and suspended in a solution of a second single chain BK analog.
  • An ultraviolet light source ⁇ e.g., Mineralight UVSL-25, Ultra Violet Products, Inc., San Gabriel, CA) is positioned 1 cm from the gently stirred suspension and irradiated in a long- wavelength range for about 10 minutes.
  • the suspension is put back on the anti-BK analog antibody affinity column and washed with a buffer containing 0.15 M NaCl, 0.1% bovine serum albumin, 0.01% polysorbate 80 and 25 KJU/ml of aprotinin to remove reaction byproducts.
  • the covalently cross-linked dimer is eluted with the same buffer system containing 0.2 M glycine or 5 M guanidine. The eluted dimer is dialyzed against buffer to remove the chaotropic agent.
  • the covalently cross- linked dimer is eluted with either 0.2 M glycine or 5 M guanidine.
  • a cleavable cross-linker non-cleavable cross-linking reagents may be utilized which contain, e.g. , alpha-hexanoate, rather than beta-ethyl- 1 ,3-dithiopropopionate moieties.
  • MSB is one example of a non-cleavable cross-linking reagent.
  • the single-chain BK analogs may be prepared by conventional solid phase peptide synthesis techniques using automated synthesis.
  • BK analogs may be prepared by recombinant DNA techniques. Based upon the known amino acid sequence of bradykinin, a synthetic gene may be constructed corresponding to that sequence, and introduced into an appropriate host by appropriate cloning vectors. Thus, it should be understood that the present invention is not merely limited to the use of BK analogs as prepared by peptide synthetic methods, but also includes the corresponding polypeptide prepared by recombinant techniques.
  • the BK analogs of the present invention inhibit ⁇ -thrombin-induced and ADP-induced platelet aggregation; block ⁇ -thrombin-induced calcium mobilization; do not block l25 I- ⁇ -thrombin binding to platelets; and prevent ⁇ - thrombin from cleaving the thrombin receptor. Protocols for the determination of these activities are set forth in Sections IIA-IIE and Section III, herein respectively.
  • Purified BK analogs may be administered in any circumstance where inhibition of thrombin-induced or ADP-induced platelet activation or platelet aggregation is sought. They are administered to subjects experiencing platelet thrombosis from any cause or they may be used prophylactically for persons undergoing surgery for insertion of artificial dacron grafts to prevent reocclusion events due to platelet thrombi. They may also be infused into individuals to prevent strokes and cerebral edema.
  • the peptides may be administered by any convenient means which will result in delivery into the bloodstream in a substantial amount.
  • Intravenous administration is presently contemplated as the preferred administration route, although intranasal administration may also be utilized. Since BK analogs are soluble in water, they may therefore be effectively administered in solution.
  • the actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, health and sex of the patient, the route of administration, and other factors.
  • An effective daily dosage of active ingredients based upon in vivo clearance studies involving HK, LK, D 3 and SEQ ID NO:20 is from about 3g per day per 70 Kg of body weight.
  • the preferred dosage is about 3g per day per 70 kg of body weight given in a single bolus infusion of 2.4 gm followed by a continuous infusion of 0.025 gm/hour.
  • Those skilled in the art should be able to derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient.
  • the amount of BK analog administered will depend upon the degree of platelet aggregation inhibition desired. While infusion of a sufficient BK analog to achieve 3g/day dosage may be advantageously utilized, more or less of the peptide may be administered as needed.
  • the therapeutic end point may be determined by monitoring platelet function by aggregation and secretion, bleeding, and vessel patency. The actual amount of the BK analog administered and the length of the therapy regime to achieve the desired intravascular concentration is readily determinable by those skilled in the art by routine methods.
  • the BK analogs may be administered in a pharmaceutical composition in a mixture with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be compounded according to conventional pharmaceutical formulation techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • the carrier will usually comprise sterile water, although other ingredients to aid solubility or for preservation purposes may be included.
  • injectable suspension may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • the preferred parenteral route of administration is intravenous administration.
  • the BK analogs may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compati ⁇ ble substances, such as sterile sodium chloride having a buffered pH compatible with physiologic conditions.
  • physiologically compati ⁇ ble substances such as sterile sodium chloride having a buffered pH compatible with physiologic conditions.
  • Such intravenous delivery vehicles are known to those skilled in the art.
  • HK was purified from plasma by a modification of the procedures of Johnson et al, Thromb. Res. 48, 187 (1987) and M ⁇ ller-Esterl et al, Methods Enzymol 163, 240 (1987).
  • One hundred ml of 1 mM DFP-treated fresh frozen plasma was thawed at 37°C, to which 10 mM benzamidine-HCl, 40 ⁇ g/ml Polybrene, 2 mM EDTA, 0.2 mM PMSF, 0.2 mg/ml soybean trypsin inhibitor, 100 U/ml aprotinin and 2 M NaCl were added according to the method of Schmaier et al, Methods in Enzymology 169, 276 (1989).
  • CM-papain-SEPHAROSE 4B equilibrated in 50 mM phosphate buffer containing 2 M NaCl, 1 mM benzamidine-HCl, 40 ⁇ g/mL Polybrene, 0.2 mM PMSF, 0.02% (w/v) NaN 3 , pH 7.5.
  • the CM-papain- SEPHAROSE 4B column was prepared by the procedure of Johnson et al, Thromb Res. 48, 187 (1987).
  • HK and LK were eluted in a single peak after the addition of a 50 mM phosphate buffer solution containing 2 mM EDTA and 0.02% (w/v) NaN* ⁇ , pH 11.5.
  • Five ml fractions were collected into tubes containing 0.25 ml of a solution consisting of 4 mM PMSF in 1 M sodium acetate, pH 4.2 to bring the final pH to 6.0.
  • the fractions containing HK and LK were then applied to a reactive Blue-Sepharose column (Sigma Chemical Corp, St. Louis, MO) equilibrated with 0.01 M sodium acetate pH 6.8 by the methods " reported by Hasan et al, J. Biol. Chem. 269, 31822 (1994).
  • LK and HK were eluted using the same buffer containing 0.3 M and 2 M NaCl, respectively.
  • HK (120 kDa) and LK (66 kDa) migrated as single bands on reduced SDS-PAGE.
  • HK reacted with monoclonal antibodies to its heavy and light chains by ELISA and western blotting, while LK was recognized only by antibodies directed at its heavy chain.
  • Purified HK retained its procoagulant activity and had a specific activity of 11-22 U/mg as previous reported by Schmaier et al, supra.
  • a number of BK analogs that encompass all or a portion of the native BK sequence (SEQ ID NO:l), including SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, and SEQ ID NO:20 and peptides SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, and SEQ ID NO: 15 were synthesized.
  • Each peptide was synthesized on an Applied Biosystems model 431 peptide synthesizer, with the carboxy-terminal amino acid covalently attached to a solid phase support, and succeeding amino acids coupled sequentially to the amino terminus.
  • the carboxyl group on the amino acid to be attached was activated with 2-(l-H-benzotriazole-l-YL)-l,l,3,3-tetramethyluroniumhexofluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt).
  • HBTU 2-(l-H-benzotriazole-l-YL)-l,l,3,3-tetramethyluroniumhexofluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • the fluorenyl-methyloxycarbonyl moiety was then attached at the amino-terminal end as a blocking group. All peptides were purified by preparative reverse-phase HPLC.
  • the individual BK analogs used to prepare the heterodimer were synthesized as described in Section LB. above.
  • a heterodimer of BK analog SEQ ID NO:20 was prepared by synthesizing SEQ ID NO:20 according to the procedure described in Section LB. above.
  • a N ⁇ -(t-butyloxycarbonyl-N ⁇ -9-fluorenylmethyloxycarbonyl-L-lysine was attached using HBTU and HOBt.
  • the amino acid was followed by attachment of N-fluorenyl-methyloxycarbonyl-L-glutamic acid- ⁇ -butyloxycarbonyl ester by the same procedure.
  • the glutamic acid's free carboxyl group was then attached to the amine side chain of the fluorenyl-methyloxycarbonyl moiety of the L-lysine resulting in a heterodimer rather than a linear amino acid.
  • BK analog SEQ ID NO:20 was then built onto the free amine of the N-fluorenyl-methyloxycarbonyl of the L-glutamic acid.
  • the heterodimer having the formula: Arg-Pro-Pro-Gly-Phe-Glu Lys-Phe-Gly-Pro-Pro-Arg was purified by reverse phase HPLC and the single species was characterized by mass spectroscopy.
  • 4-MAP Four Branch MAP of BK analog SEQ ID NO:20, hereinafter called "4-MAP,” was prepared.
  • MAP is an acronym for "multiple antigenic peptide”.
  • the structure for 4-MAP is as follows:
  • 4-MAP a resin core, having a ⁇ -alanine attached through its carboxyl group, was joined to a free carboxyl of lysine through the free amine of ⁇ -alanine ( ⁇ Ala) to form a lysine- ⁇ -alanine complex.
  • ⁇ Ala free amine of ⁇ -alanine
  • Two additional lysine residues were then attached by their free amine groups to the free carboxyl of the first lysine.
  • Four molecules of SEQ ID NO:20 were then attached through their phenylalanine residues to the free amino groups of the two lysine residues, following activation with HBTU and HOBt as described in Section I.D. above.
  • the 4-MAP was purified by reverse phase HPLC and then characterized by mass spectroscopy.
  • ⁇ -thrombin [0.125 U/ml (1 nM) final concentration] was then added to initiate platelet activation. Stirred platelets were allowed to incubate with ⁇ -thrombin and additions for 1 min. In other experiments, platelets were stimulated with TRAP (0.625 to 2.5 ⁇ M), ADP (1-5 ⁇ M)(Sigma), collagen (1.25 ⁇ g/ml) (Horm, Kunststoff, Germany), or U-46619 (1 ⁇ M)(Calbiochem Behring, San Diego, CA). Additional experiments were performed with washed platelets stimulated with ⁇ -thrombin (1 nM) in the presence of human fibrinogen (100 mg/dl).
  • ⁇ -thrombin and human fibrinogen were purchased from Enzyme Research Laboratories, South Bend, IN.
  • the entire platelet sample was centrifuged at 10,900 xg (Model E, Beckman Instruments, Palo Alto, CA) over a 0.135 mM formaldehyde, 5 mM EDTA solution (1 part of formaldehyde-EDTA to 4 parts of platelet suspension) and stored on ice until an aliquot of the supernatant was assayed for 5-[ 14 C]hydroxytryptamine secretion.
  • Percent secretion was determined by the ratio of the loss of 5-[ l4 C]hydroxytryptamine in the supernatant of the agonist-treated specimen to the loss of 5-[ ,4 C]hydroxytryptamine in the supernatant of the platelet lysate after the value of the control supernatant (i.e., the level of 5- [ 14 C]hydroxytryptamine in a unstimulated sample) was subtracted from both.
  • each peptide that contained the amino acid sequence Arg-Pro-Pro-Gly-Phe produced concentration- dependent inhibition of ⁇ -thrombin-induced platelet aggregation and secretion.
  • the degree of inhibition of platelet aggregation was greater at a given concentration of peptide than the degree of inhibition of platelet secretion.
  • the most potent thrombin inhibitor among those tested was the BK analog SEQ ID NO: 14, which inhibited platelet aggregation and secretion with an IC 50 of 0.23 and 0.5 mM, respectively (Table I).
  • BK (SEQ ID NO:l) is also a potent inhibitor of ⁇ -thrombin-induced platelet activation with an IC 50 of 0.25 mM and 1.0 mM for aggregation and secretion inhibition, respectively.
  • the BK analog comprising the five amino terminal amino acids of the native BK sequence segment, Arg-Pro-Pro-Gly-Phe (SEQ ID NO:20), inhibited ⁇ -thrombin- induced platelet aggregation with an IC 50 of 0.5 mM.
  • SEQ ID NO:20 inhibited 95% of platelet aggregation and 25% of secretion, while two scrambled peptides having the amino acid SEQ ID NO:20, SEQ ID NO:5 and SEQ ID NO:9, did not inhibit ⁇ -thrombin-induced platelet aggregation and secretion at 1 mM.
  • BK analogs of the mid or carboxy terminal regions of BK, SEQ ID NO:7 and SEQ ID NO: 16 were poor inhibitors of ⁇ -thrombin-induced platelet activation with IC 50 > 2 mM.
  • BK analogs inhibited ⁇ -thrombin-induced (1 nM) platelet activation in the presence of 100 mg/dl human fibrinogen and in platelet-rich plasma.
  • SEQ ID NO: 16 >3.0 mM >2.0 mM
  • the cytoplasmic free Ca 2+ concentration ([Ca 2+ ] z ) was measured using the fluorescent Ca 2+ indicator fura-2 (Molecular Probes, Inc., Eugene, OR).
  • Gel filtered platelets in Hepes-Tyrode's buffer were loaded with fura-2 by incubation at 37°C with 1 ⁇ M fura-2/acetoxymethyl ester for 45 min according to the method of Rasmussen et al, J. Biol Chem. 268, 14322 (1993).
  • the labeled platelets were then re-gel filtered to remove any excess probe.
  • the minimum emission was determined on a 20 mM digitonin, 10 mM EGTA solubilized platelet sample; maximum emission was determined on the same sample with 10 mM Ca + added.
  • the intraplatelet free Ca 2+ concentration was calculated by the method of Grykiewicz et al, J. Biol. Chem. 260, 3440 (1985).
  • the intraplatelet free Ca 2+ concentration was calculated by the method of Grykiewicz et al, J. Biol. Chem. 260, 3440 (1985).
  • the K ⁇ for fura- 2 was assumed to be 224 nM.
  • thrombin alone induces a substantial change in Ca 2+ mobilization which was inhibited by HK ( Figure 2B).
  • BK and BK analog SEQ ID NO: 14 block ⁇ -thrombin-induced calcium mobilization similar to their parent protein, HK ( Figure 2C and Figure 2D).
  • Increasing concentrations of BK and BK analog SEQ ID NO: 14 produced decreasing Ca 2+ mobilization with an IC 50 of 0.23 and 0.3 mM, respectively.
  • the results of the concentration dependent study are presented in Figure 3.
  • BK analogs described herein inhibited iodinated ⁇ -thrombin binding to platelets.
  • Gel filtered platelets were placed into polypropylene tubes and diluted with Hepes-Tyrode's buffer, containing 2 mM CaCl 2 and 50 ⁇ M ZnCl 2 and additions, to a final concentration of 2 X 10 s platelets/ml.
  • the reaction was started by the addition of 1 nM 125 I- ⁇ -thrombin, which was prepared by using the iodogen technique as reported by Meloni et al, J. Biol. Chem. 266, 6786 (1991). Incubations were performed at 37°C for specified times with various additions.
  • SPAN 12 is an antibody to the thrombin receptor on platelets, which is specific for such an epitope. Studies were also performed to determine the effect of BK analogs on an epitope recognized by monoclonal antibody ATAP138. The antibody is directed to an epitope on the thrombin receptor which is preserved after ⁇ - thrombin cleaves the receptor ( Figures 7A-7D).
  • Monoclonal antibody SPAN 12 was reared to the 12 amino acids, Asn- Ala-Thr-Leu- Asp-Pro- Arg-Ser-Phe-Leu-Leu- Arg (SEQ ID NO:2), that bridge the ⁇ -thrombin cleavage site on the thrombin receptor by the methods of Molinot et al, J. Biol Chem. 270:In Press, 1995.
  • Monoclonal antibody ATAP138 recognizes the epitope Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SEQ ID NO:3) on the thrombin receptor which is preserved after cleavage by ⁇ -thrombin as reported by Brass et al, J.
  • Platelets for flow cytometry studies were prepared from 53.3 ml fresh blood anticoagulated with 8.7 ml acid citrate dextrose (10 mM trisodium citrate, 66 mM citric acid, 111 mM glucose, pH 4.6). Washed platelets from platelet-rich plasma were prepared by centrifugation at 180 x g for 15 min. at room temperature. The platelet-rich plasma was brought to a final concentration of 2.8 ⁇ M with PGE1 (Sigma) and 1:25 (vol:vol) with 1 M sodium citrate. After a 5 min. incubation at room temperature, the platelet-rich plasma was centrifuged at 1200 x g for 10 min. at room temperature.
  • the platelet pellet was then re- suspended in 10 ml of platelet wash buffer (128 mM NaCl, 4.26 mM NaH 2 PO 4 , 7.46 mM Na 2 HPO 4 , 4.77 mM sodium citrate, 2.35 mM citric acid, 5.5 mM glucose, 3.5 mg/ml bovine serum albumin, pH 6.5) followed by centrifugation at 1200 x g for 5 min. at room temperature.
  • platelet wash buffer (128 mM NaCl, 4.26 mM NaH 2 PO 4 , 7.46 mM Na 2 HPO 4 , 4.77 mM sodium citrate, 2.35 mM citric acid, 5.5 mM glucose, 3.5 mg/ml bovine serum albumin, pH 6.5
  • ⁇ -thrombin (0.125 U/ml or 1 nM).
  • Primary antibodies were added at a final concentration of 2 ⁇ g/ml and the antibodies were incubated with the platelets for 30 min at 4°C. After incubation, the platelets were diluted with 500 ⁇ l of platelet suspension buffer and again centrifuged at 1200 x g for 5 min. at room temperature. The platelet pellets were then re-suspended in 100 ⁇ l of platelet suspension buffer and incubated with a 1:40 dilution of an anti- mouse IgG conjugated with FITC. After an additional incubation for 30 min. at 4°C, the platelets were again centrifuged at 1200 x g for 5 min. followed by re- suspension in 500 ⁇ l of platelet suspension buffer.
  • Mouse IgG and an antibody to the epitope CD62 were used as controls.
  • Mouse IgG (Code #4350) was purchased from BioSource, Camarillo, CA.
  • the fluorescence of bound FITC-anti-IgG to platelets was monitored on an Epics-C flow cytometer (Coulter Electronics, Hialeah, FL). Light scatter and fluorescence channels were set at logarithmic gain. Laser excitation was at 488 nm. Green fluorescence was observed through a 525 nm band pass filter. The relative fluorescence intensity of at least 15,000 platelets was analyzed in each sample.
  • An antibody to CD62 (P-selectin) was purchased from Becton-Dickinson (Catalogue # 550014), San Jose, CA.
  • SPAN 12 detects an antigen on the thrombin receptor on unstimulated platelets.
  • a thrombin receptor was described by Vu et al, Cell 64, 1057 (1991).
  • Figure 6A solid curve
  • cleavage study was performed according to the method of Molino et al. , J. Biol. Chem. 270, 11168 (1995), in which NATl 2 (SEQ ID NO:2) was dissolved in a solution of 0.01 M NaH 2 PO 4 and 0.15 M NaCl, pH 7.4. The mixture was then incubated with 8 nM ⁇ -thrombin for one hour at 37° C either
  • each mixture was separated by applying the mixture to a Vyadec C-18 HPLC column in 0.1% trifluoroacetic acid and eluting the mixture with a gradient from 0% to 100% of 80% MeCN and 0.1% trifluoroacetic acid and eluting the mixture with a gradient from 0% to 100% of 80% MeCN and 0.1 % trifluoroacetic acid.
  • the size of the separated products were confirmed by mass spectrometry.
  • NAT 12 As shown in Figure 8 A, NAT 12 (SEQ ID NO:2) when measured by HPLC, produced a single peak. Peak 1 of Figure 8A represents 100%.
  • NAT12 SEQ ID NO:2
  • Figure 8C peak 1
  • peaks 3 and 2 shown in Figure 8C
  • peak 1 of NATl 2 SEQ ID NO:2
  • ⁇ - thrombin BK analog SEQ ID NO:20
  • FIG. 8D The cleavage products of NATl 2 ( Figure 8D, peaks 3 and 2) constitute 31 % and 26%, respectively, of the non-treated peak area ( Figure 8A).
  • Figure 8B represents the chromatograph for isolated BK analog SEQ ID NO:20.
  • NAT12 SEQ ID NO:2
  • ⁇ -thrombin reduced the size of the original peak for NAT12 (SEQ ID NO:2) ( Figure 8A) by only 32% ( Figure 8E).
  • the two ⁇ -thrombin cleavage fragments peaks 3 and 2 in Figure 8E, constituted only 18% and 14%, respectively, of the area of peak 1 in Figure 8 A.
  • the fourth peak seen in Figure 8E represented a peak from the HK preparation and is not an additional ⁇ -thrombin cleavage fragment.
  • stage III surgical anesthesia was maintained with 20 mg/ml of intravenous pentobarbital.
  • a carotid artery and a jugular vein were then exposed.
  • a catheter was inserted into the exposed carotid artery for withdrawal of blood samples and monitoring the animal's blood pressure (Gould, Inc., Cardiovascular Products, Oxnard, CA).
  • a catheter was inserted into the exposed jugular vein for administering the anesthetic and BK analog SEQ ID NO:20.
  • BK analog SEQ ID NO:20 For the clearance study, a single bolus of BK analog SEQ ID NO:20 was injected. The amount of BK analog SEQ ID NO:20 injected was calculated from the weight of the animal such that the blood volume was 1 mM with peptide. For example: for a 2.5 kg rabbit, 7% of its weight gives an estimated blood volume of 175 ml. Accordingly, 89 mg of BK analog was injected to make the 175 ml plasma sample ImM. Depending upon the size of the animal, 75 to 90 mg peptide was injected. Blood samples were collected at 2, 4, 6, 8, 10, 20, 30, 40, 60, 90, and 120 minute intervals after infusion into a 0.013 M sodium citrate anticoagulant solution.
  • Plasma was prepared from each of the blood samples collected over time by centrifugation of the blood samples at 10,00 xg for two minutes. Aliquots of the plasmas were assayed for the presence of the BK analog SEQ ID NO:20 antigen by the ELISA technique using a MARKIT-M [1-5] BK assay from Dainippon Pharmaceutical Co., Ltd., Osaka, Japan.
  • Platelet aggregation studies on the PRP were conducted on a 4- channel aggregometer (BioData-PAP-4, Bio Data Corp., Hatboro, PA). The degree of platelet aggregation was determined by measuring the increase in light transmission through a stirred suspension of PRP maintained at 37°C. Platelet aggregation was induced in the PRP sample by addition of 20 ⁇ M ADP and ⁇ - thrombin according to the method of Harfenist et al, Thromb. Haemost. 53, 183 (1985). Gamma-thrombin (Enzyme Research Laboratories, South Bend, IN) was used for this study in lieu of ⁇ -thrombin because it does not proteolyze fibrinogen and clot platelet-rich plasma.
  • rabbit platelets display a variable response to ⁇ -thrombin.
  • Each rabbit's platelets were evaluated before BK analog infusion for their threshold response to ⁇ -thrombin.
  • the rabbit platelets used in this experiment were responsive to 10 nM to 40 nM ⁇ -thrombin.
  • Simultaneous ⁇ -thrombin-induced platelet aggregation studies were performed with 10, 20, and 40 nM ⁇ -thrombin and 20 ⁇ M ADP.
  • the peak plasma concentration of BK analog SEQ ID NO:20 after infusion was 60 mg/ml (0.120 mM) for two of three rabbits, as determined by ELISA. No unfavorable effects were observed in the animals following the bolus injection of the BK analog.
  • the rabbits' blood pressure, pulse, and platelet count remained stable and there was no abnormal bleeding at the surgical sites of cutdowns and intubations.
  • the half-life of BK analog SEQ ID NO:20 antigen clearance in plasma was calculated to be 6.6 minutes after infusion. Clearance of BK analog SEQ ID NO:20 initially was not due to renal excretion, as ligating the animal's renal arteries did not lengthen the half-life of the agent ( Figure 9, Rabbit 2). Therefore, the major determinant of the immediate clearance of the BK analog SEQ ID NO:20 antigen was attributed to binding and/or metabolism.
  • BK analog SEQ ID NO:20 had a prolonged biologic clearance. After a single bolus infusion of BK analog SEQ ID NO:20, 10 nM ⁇ -thrombin-induced platelet aggregation was inhibited 100% for over 4 hours (data not shown), 20 nM ⁇ -thrombin-induced platelet aggregation was inhibited >50% for 2.75 hours, and 40 nM ⁇ -thrombin-induced platelet aggregation was inhibited >50% for one hour. The data further indicated that there was >50% " inhibition of ADP-induced platelet aggregation for roughly 45 minutes. This latter finding suggested that thrombin mediates ADP-induced platelet activation in vivo as well.
  • Plasma samples were obtained from normal human volunteers. Platelet counts were measured with a Coulter counter, Model 2F (Coulter, Hialeah, FL) and adjusted to a platelet count of 200,000 platelets/ ⁇ l. Each individual's platelets at baseline were measured for their threshold response to ⁇ -thrombin. Typical threshold levels were between 10 nM to 40 nM.
  • Figure 11 shows the results of using a heterodimer BK analog (labeled "HETERODIMER”) and 4-MAP on ⁇ -thrombin-induced platelet activation.
  • Human platelets in PRP were treated with 20 nM ⁇ -thrombin.
  • Figure 11 shows the tracings from the aggregometer.
  • 1 mM BK analog SEQ ID NO:20, 0.05 mM 4-MAP, or 0.5 mM of the heterodimer () was reacted with 20 nM ⁇ -thrombin, the aggregation tracing was abolished.
  • the specificity of this reaction was demonstrated by comparing the results to those for a reaction done with 1 mM of a non-BK analog peptide (SEQ ID NO:22).
  • SEQ ID NO:22 was unable to alter the ability of ⁇ -thrombin to induce platelet activation.

Abstract

L'administration d'un peptide analogue lié à une séquence de brakykinine permet d'inhiber l'activation des plaquettes et des autres cellules induite par la thrombine et par l'adénosine diphosphate sans modifier l'autre activité protéolytique de la thrombine. Les analogues de la brakykinine sont des peptides qui ont subi des substitutions, des adjonctions ou des suppressions par rapport aux quatre premiers acides aminés de la séquence d'acides aminés de la bradykinine native. Les analogues de la bradykinine selon l'invention inhibent l'activation et la sécrétion des plaquettes induites par l'α-thrombine et l'adénosine diphosphate, et inhibent également la mobilisation du calcium induite par l'α-thrombine. Par ailleurs, ils empêchent l'α-thrombine de diviser son récepteur de plaquettes. Les analogues de la bradykinine peuvent comprendre des peptides à simples et à multiples chaînes.
PCT/US1996/009940 1995-06-09 1996-06-07 Analogues de la bradykinine utilises comme inhibiteurs selectifs de la thrombine WO1996041640A1 (fr)

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DE69627191T DE69627191T2 (de) 1995-06-09 1996-06-07 Bradikininanaloge als selektive thrombininhibitoren
AT96923268T ATE235912T1 (de) 1995-06-09 1996-06-07 Bradikininanaloge als selektive thrombininhibitoren
US08/676,242 US6143719A (en) 1995-06-09 1996-06-07 Bradykinin analogs as selective thrombin inhibitors
AU63828/96A AU703256B2 (en) 1995-06-09 1996-06-07 Bradykinin analogs as selective thrombin inhibitors
EP96923268A EP0871464B1 (fr) 1995-06-09 1996-06-07 Analogues de la bradykinine utilises comme inhibiteurs selectifs de la thrombine
JP50324397A JP4067117B2 (ja) 1995-06-09 1996-06-07 選択性スロンビン抑制剤としてのブラジキニン同族体
HK99101383A HK1016090A1 (en) 1995-06-09 1999-04-07 Bradykinin analogs as selective thrombin inhibitors

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WO2000035407A2 (fr) * 1998-12-16 2000-06-22 Temple University - Of The Commonwealth System Of Higher Education Inhibition de l'angiogenese par des analogues de peptides du domaine 3 du kininogene a poids moleculaire eleve
EP1019070A1 (fr) * 1997-04-23 2000-07-19 The Regents Of The University Of Michigan Analogues de bradykinine inhibiteurs selectifs de l'activation cellulaire
US6284726B1 (en) 1998-11-10 2001-09-04 Temple University - Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by peptide analogs of high molecular weight kininogen domain 5
EP1212352A1 (fr) * 1999-08-17 2002-06-12 Thromgen, Inc. Analogues peptidiques comme inhibiteurs selectifs de l'activation par la thrombine du recepteur 1 active par une protease
AU761624B2 (en) * 1997-04-23 2003-06-05 Regents Of The University Of Michigan, The Bradykinin analogs as selective inhibitors of cell activation
US6767889B1 (en) 1998-11-10 2004-07-27 Temple University Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by high molecular weight kininogen and peptide analogs thereof
US6869931B1 (en) 1998-12-16 2005-03-22 Temple University - Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by high molecular weight kininogen domain 3 peptide analogs
US6982249B1 (en) 1997-04-23 2006-01-03 The Regents Of The University Of Michigan Bradykinin analogs as selective inhibitors of cell activation
US6994852B1 (en) 1999-11-12 2006-02-07 Temple University-Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by antibodies against high molecular weight kininogen domain 5
US10299714B2 (en) 2010-12-02 2019-05-28 Becton, Dickinson And Company Blood collection devices containing blood stabilization agent including variegin or analog thereof and/or a polysulfated disaccharide

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6346510B1 (en) 1995-10-23 2002-02-12 The Children's Medical Center Corporation Therapeutic antiangiogenic endostatin compositions
US20020076782A1 (en) * 1996-07-05 2002-06-20 John P.N. Rosazza Purified nitric oxide synthase
US7074765B2 (en) * 2003-05-01 2006-07-11 The Regents Of The University Of Michigan Synthetic peptide analogs of Arg-Pro-Pro-Gly-Phe as selective inhibitors of thrombin and thrombin activation of protease activated receptors 1 and 4
WO2006023844A2 (fr) * 2004-08-20 2006-03-02 Entremed, Inc. Compositions et procedes comportant des antagonistes de recepteur active par la proteinase
WO2006045503A1 (fr) 2004-10-19 2006-05-04 Lonza Ag Procede de synthese de peptides en phase solide
US7879792B2 (en) * 2005-06-02 2011-02-01 The Regents Of The University Of Michigan Synthetic peptide inhibitors of thrombin and thrombin activation of protease activated receptors 1 and 4

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4693993A (en) * 1985-06-13 1987-09-15 Stewart John M Bradykinin antagonist peptides
US4870017A (en) * 1985-09-20 1989-09-26 Cetus Corporation Bacterial methionine N-terminal peptidase
US4923963A (en) * 1987-09-02 1990-05-08 Nova Technology Limited Partnership Bradykinin antagonist peptides
US5231080A (en) * 1985-10-15 1993-07-27 Hoechst Aktiengesellschaft Method for the treatment of atherosclerosis, thrombosis, and peripheral vessel disease
US5385889A (en) * 1991-04-19 1995-01-31 Scios Nova Inc. Bradykinin antagonist peptides
US5409899A (en) * 1992-01-17 1995-04-25 Adir Et Compagnie Pseudopeptide compounds having anti-inflammatory activity
US5416191A (en) * 1991-04-01 1995-05-16 Cortech, Inc. Bradykinin antagonists

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8305985D0 (en) * 1983-03-04 1983-04-07 Szelke M Enzyme inhibition
US5084274A (en) * 1987-11-17 1992-01-28 Scripps Clinic And Research Foundation Inhibition of arterial thrombotic occlusion or thromboembolism
US5202421A (en) * 1988-12-27 1993-04-13 Mochida Pharmaceutical Co., Ltd. Anticoagulant substance obtained from urine and process for the preparation thereof
CZ203693A3 (en) * 1991-04-01 1994-07-13 Cortech Bradykinin antagonists
AU2548992A (en) * 1991-08-13 1993-03-16 Temple University - Of The Commonwealth System Of Higher Education Modulation of blood pressure and inhibition of platelet activation with kininogen fragment
EP0632829A1 (fr) * 1992-03-02 1995-01-11 Biogen, Inc. Antagonistes de recepteur de thrombine
IL107400A0 (en) * 1992-11-10 1994-01-25 Cortech Inc Bradykinin antagonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4693993A (en) * 1985-06-13 1987-09-15 Stewart John M Bradykinin antagonist peptides
US4870017A (en) * 1985-09-20 1989-09-26 Cetus Corporation Bacterial methionine N-terminal peptidase
US5231080A (en) * 1985-10-15 1993-07-27 Hoechst Aktiengesellschaft Method for the treatment of atherosclerosis, thrombosis, and peripheral vessel disease
US4923963A (en) * 1987-09-02 1990-05-08 Nova Technology Limited Partnership Bradykinin antagonist peptides
US5416191A (en) * 1991-04-01 1995-05-16 Cortech, Inc. Bradykinin antagonists
US5385889A (en) * 1991-04-19 1995-01-31 Scios Nova Inc. Bradykinin antagonist peptides
US5409899A (en) * 1992-01-17 1995-04-25 Adir Et Compagnie Pseudopeptide compounds having anti-inflammatory activity

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts Service (C A S); 22 December 1980 (1980-12-22), IMAI S, MATSUBARA I: "Effects of Prostacyclin on Platelet Aggregation as Studied with 'Filter-Loop' Technique in the Flowing Blood of the Dog", XP002940105, Database accession no. 93-231243T *
Chemical Abstracts Service (C A S); 24 May 1976 (1976-05-24), ISHIKAWA Y, ET AL: "Prostaglandin Synthetase Activity and Hormone Responsiveness in Normal and SV40 Transformed WI-38 Fibroblasts", XP002940104, Database accession no. 84-148985E *
Chemical Abstracts Service (C A S); 26 October 1987 (1987-10-26), ALHEID U, FROELICH J C, FOERSTERMANN U: "Endothelium-Derived Relaxing Factor from Cultured Human Endothelial Cells Inhibits Aggregation of Human Platelets", XP002940106, Database accession no. 107-148141V *
Chemical Abstracts Service (C A S); 28 August 1989 (1989-08-28), LOEB A L, PEACH M J, GEAR A R L: "Endothelium-Dependent Potentiation of Human Platelet Aggregation", XP002940107, Database accession no. 111-71858G *
CIRCULATION, vol. 94, no. 3, 1996, pages 517 - 528 *
DATABASE CAPLUS [online] HASAN ET AL: "Bradykinin and its Metabolite, Arg-Pro-Pro-Gly-Phe, are Selective Inhibitors of alpha-Thrombin-Induced Platelet Activation", XP002940103, accession no. STN Database accession no. 1996:519830 *
MARTIN T W, MICHAELIS K C: "BRADYKININ STIMULATES PHOSPHODIESTERATIC CLEAVAGE OF PHOSPHATIDYLCHOLINE IN CULTURED ENDOTHELIAL CELLS", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 157, no. 03, 30 December 1988 (1988-12-30), US, pages 1271 - 1279, XP002940609, ISSN: 0006-291X, DOI: 10.1016/S0006-291X(88)81012-X *
STEWART J M, VAVREK R J: "BRADYKININ CHEMISTRY: AGONISTS AND ANTAGONISTS", ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY, SPRINGER, US, vol. 156A, no. 03, 1 January 1979 (1979-01-01), US, pages 585 - 589, XP002940613, ISSN: 0065-2598 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6982249B1 (en) 1997-04-23 2006-01-03 The Regents Of The University Of Michigan Bradykinin analogs as selective inhibitors of cell activation
AU761624B2 (en) * 1997-04-23 2003-06-05 Regents Of The University Of Michigan, The Bradykinin analogs as selective inhibitors of cell activation
EP1019070A1 (fr) * 1997-04-23 2000-07-19 The Regents Of The University Of Michigan Analogues de bradykinine inhibiteurs selectifs de l'activation cellulaire
EP1019070A4 (fr) * 1997-04-23 2001-05-16 Univ Michigan Analogues de bradykinine inhibiteurs selectifs de l'activation cellulaire
US6767889B1 (en) 1998-11-10 2004-07-27 Temple University Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by high molecular weight kininogen and peptide analogs thereof
US6284726B1 (en) 1998-11-10 2001-09-04 Temple University - Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by peptide analogs of high molecular weight kininogen domain 5
WO2000035407A3 (fr) * 1998-12-16 2000-09-08 Univ Temple Inhibition de l'angiogenese par des analogues de peptides du domaine 3 du kininogene a poids moleculaire eleve
AU774694B2 (en) * 1998-12-16 2004-07-01 Temple University - Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by high molecular weight kininogen domain 3 peptide analogs
US6869931B1 (en) 1998-12-16 2005-03-22 Temple University - Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by high molecular weight kininogen domain 3 peptide analogs
WO2000035407A2 (fr) * 1998-12-16 2000-06-22 Temple University - Of The Commonwealth System Of Higher Education Inhibition de l'angiogenese par des analogues de peptides du domaine 3 du kininogene a poids moleculaire eleve
EP1212352A1 (fr) * 1999-08-17 2002-06-12 Thromgen, Inc. Analogues peptidiques comme inhibiteurs selectifs de l'activation par la thrombine du recepteur 1 active par une protease
EP1212352A4 (fr) * 1999-08-17 2002-12-04 Thromgen Inc Analogues peptidiques comme inhibiteurs selectifs de l'activation par la thrombine du recepteur 1 active par une protease
US6544750B1 (en) * 1999-08-17 2003-04-08 Thromgen, Inc. Peptide analogs as selective inhibitors of thrombin activation of protease activated receptor 1
US6994852B1 (en) 1999-11-12 2006-02-07 Temple University-Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by antibodies against high molecular weight kininogen domain 5
US7332161B2 (en) 1999-11-12 2008-02-19 Temple University - Of The Commonwealth System Of Higher Education Treatment of disease with antibodies against high molecular weight kininogen domain 5
US10299714B2 (en) 2010-12-02 2019-05-28 Becton, Dickinson And Company Blood collection devices containing blood stabilization agent including variegin or analog thereof and/or a polysulfated disaccharide

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EP0871464A1 (fr) 1998-10-21
EP0871464B1 (fr) 2003-04-02
ATE235912T1 (de) 2003-04-15
AU703256B2 (en) 1999-03-25
DE69627191D1 (de) 2003-05-08
CA2221865C (fr) 2007-12-11
DE69627191T2 (de) 2004-01-08
JP2001511762A (ja) 2001-08-14
US6143719A (en) 2000-11-07
HK1016090A1 (en) 1999-10-29
AU6382896A (en) 1997-01-09
JP4067117B2 (ja) 2008-03-26
EP0871464A4 (fr) 2001-05-16
CA2221865A1 (fr) 1996-12-27

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